Contents lists available at ScienceDirect Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology Tumor microenvironment and Oral Squamous Cell Carcinoma: A crosstalk between the infammatory state and tumor cell migration Alessandro Alves a , Leonardo Diel b , Grasieli Ramos c , Antônio Pinto d , Lisiane Bernardi e , John Yates 3rd f , Marcelo Lamers e, ⁎ a School of Dentistry, University Center Univates, Lajeado, RS, Brazil b School of Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil c School of Dentistry, University of Oeste de Santa Catarina, Joaçaba, SC, Brazil d Clayton Foundation Peptide Biology Lab, Salk Institute for Biological Studies, United States e Department of Morphological Sciences, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil f Department of Molecular Medicine, The Scripps Research Institute, United States ARTICLEINFO Keywords: Oral cancer Cell migration Rho GTPases Interleukins IL-6 ABSTRACT Objectives: To analyze the infammatory millieu in oral squamous cell carcinoma (OSCC) tumors and the in- fuence of macrophages related-cytokines on the tumor cell migration. Materials and methods: Infammatory protein profleandmacrophagepopulation(M2/M1ratio)ofhumanOSCC fragments were analyzed by proteomic analysis and fowcytometryassayrespectively.Toevaluatetheefectsof infammation on OSCC behavior, we analyzed the role of polarized macrophages and cytokines (IL-6, IL-1β and TNF-α) on OSCC cell lines (SCC25 and Cal27) responsiveness by western blotting (cell signaling) and time-lapse (cell migration). Also, it was addressed the crosstalk of IL-6-STAT3 axis with cell migration signaling using a STAT3 inhibitor (Stattic®) and a pull down assay for the RhoGTPase Rac1 activity. Results: It was observed a ~2 fold predominance of M2 over M1 macrophages and a pro-infammatory state in OSCC fragments. The M2 conditioned media increased migration speed and directionality of highly invasive OSCC cells (SCC25). OSCC cell lines were responsive to cytokine stimuli (IL6, IL-1β and TNF-α), but only IL-6 increasedmigrationpropertiesofOSCCcells.ThisefectwasdependentonSTAT3-phosphorylationlevels,which interfered with Rac1 activation levels. Conclusion: Ourresultssuggestthattheinfammatorymilieumight favorinvasionandmetastasisofOSCCbythe direct efect of macrophage-related cytokines on tumor migration. Introduction Tumor microenvironment (TME) of Oral Squamous Cell Carcinoma (OSCC) is composed by reprogrammed stromal cells [1] that produce cytokines that may promote neoplastic cells growth and invasion of tissues [2,3]. TME is usually characterized by a chronic infammatory state with the presence of immunosuppressive cells with pro-tumor activity: regulatory T lymphocytes (Treg cells), M2 macrophages, N2 neutrophils and myeloid-derived suppressor cells [4,5]. In this system, there is an intense cellular communication through releasing of in- fammatory cytokines, such as IL-6, TNF-α and IL-1β [5,6]. TME re- presents a challenge for the treatment of OSCC patients and a better understanding of these interactions can lead to the development of more efective therapies [7]. Among TME cells, tumor-associated macrophages (TAMs) are the most infuential in tumor progression and their presence is related to a worse prognosis [1,8,9]. One of the main characteristics of macro- phages is their plasticity, since they acquire diferent phenotypes ac- cording to signals from the surrounding microenvironment. Typically macrophages are divided into two main groups: M1 and M2 [10,11]. Macrophage M2 is related to proliferation and survival of tumor cells, increase of angiogenesis, invasion of adjacent tissues and metastasis and promotion of epithelial-mesenchymal transition (EMT). Also, M2 macrophages are associated to lower survival in several tumors, in- cluding OSCC [12–15]. During EMT, cancer cells modify their cytoskeleton and shape, lose epithelial markers (e-cadherin) and earn mesenchymal markers (n- cadherin), leading to increased cellular motility [16]. EMT may be https://doi.org/10.1016/j.oraloncology.2020.105038 Received 28 May 2020; Received in revised form 18 September 2020; Accepted 4 October 2020 ⁎ Corresponding author at: Department of Morphological Sciences, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Rua Sarmento Leite, 500, Porto Alegre, RS CEP 90050-170, Brazil. E-mail addresses: leocvr@bol.com.br (L. Diel), jyates@scripps.edu (J. Yates), marcelo.lamers@ufrgs.br (M. Lamers). Oral Oncology 112 (2021) 105038 1368-8375/ © 2020 Elsevier Ltd. All rights reserved. T